Venoms of the marine cone snail of the genus Conus are a rich and extremely diverse source of bioactive components. With more than 800 species of Conus available worldwide, cone snail venoms appear as one of the richest source of naturally occurring peptides exhibiting a wide array of biological activity. The conopeptides target numerous and various molecular entities including voltage-sensitive ion channels, ligand-gated ion channels and G-protein-coupled receptors, with high affinity and specificity (McIntosh et al., 1999; Olivera et al., 1985; Olivera et al., 1990). Among all existing conopeptides, only a minority has been extensively characterized from isolation, primary structure elucidation to precise molecular target identification. However, increasing attention has been brought to this research area as conopeptides provide new and important tools for dissecting the function of previously uncharacterised channels. This also allows opportunities for entirely new biomedical application with the use of new drugs acting on original physiological targets. This can be exemplified by the discovery and use of omega-conotoxins for differentiating particular calcium subtypes and the further use of one of them as a drug (Prialt®) in pain management (Kerr and Yoshikami, 1984; Olivera et al., 1984; Olivera et al., 1987).
The publication of the first representatives of the mu-conopeptide family occurred in 1983 with the characterization of the geographutoxins exhibiting a myotoxic activity (Sato et al., 1983). This was followed by the isolation and identification of several other mu-conopeptides since then. To date, a total of 9 mu-conopeptides have been so far characterized from 6 different cone snail species, including mainly piscivorous species and one molluscivorous species.
All these mu-conopeptides display a common primary structure demonstrated by the conserved position of the cysteine residues in the sequence. The disulfide bonding is between Cys1-Cys4, Cys2-Cys5 and Cys3-Cys6. This fold leads to a constrained tertiary structure that has been studied for several representatives of the mu-conopeptide family (Hill et al., 1996; Keizer et al., 2003; Nielsen et al., 2002; Ott et al., 1991; Wakamatsu et al., 1992). It has been demonstrated in numerous studies that the mu-conopeptides target more or less specifically various voltage-sensitive sodium channels (Becker et al., 1989; Bulaj et al., 2005; Cruz et al., 1985; Cruz et al., 1989; Fainzilber et al., 1995; French et al., 1996; Safo et al., 2000; Sato et al., 1991; West et al., 2002). Whatever the subtype of sodium channels targeted, the pharmacological effect always consists in a blockade of the channel conductance leading to an inhibition of the voltage-sensitive channel functionality.
Voltage-sensitive sodium channels (VSSCs) are transmembrane proteins fundamental for cell communication as they generate action potentials and enable its propagation in most vertebrate and invertebrate excitable cells. Presently 9 genes have been identified that code for mammalian VSSCs (Yu and Catterall, 2003). VSSCs are classified according to their sensitivity to tetrodotoxin (TTX), a toxin isolated in particular from the puffer fish. VSSCs blocked by TTX are known as TTX-sensitive, while the others are TTX-resistant channels. Each subtype of VSSC has a specialised function depending on its cellular and tissue localization.
VSSCs have a major role in the transmission of the action potential in muscles as well as in nerves, thus providing a key target in anaesthesia. Drugs such as lidocaine or procaine act through the inhibition of VSSCs present in sensory fibres (Scholz, 2002). However, inhibition does not occur equally in all fibres due to the presence of numerous VSSCs subtypes differently affected by the drugs. Among them, TTX-resistant VSSC subtypes have a predominant role in the transmission of pain and are currently not specifically targeted by any known drug. Furthermore, the short duration of time of lidocaine and procaine as well as the well-documented side-reactions or allergy in response to their application make them difficult to use as anaesthesics in specific cases. In this context, compounds allowing specific inhibition of TTX-resistant VSSCs would appear as a major achievement for pain control. As an example, the subtype Nav1.8 contributes to the initiation and maintenance of hyperalgesia. In early stages of neuropathic pain, the expression of Nav1.8 is reduced in the primary afferent neurones which are injured, while expression levels of Nav1.8 are maintained in adjacent neurones (Decosterd et al., 2002; Gold et al., 2003). However, two days following sciatic nerve injury there is a significant upregulation of Nav1.8 expression as well as a proportional increase in the TTX-resistant compound action potential, at a conduction velocity consistent with C fibres (Gold et al., 2003). This strongly supports an important role for Nav1.8 in neuropathic pain.
The VSSCs thus represent useful targets which inhibition or modulation allow anaesthesia, analgesia and pain control (Baker and Wood, 2001; Julius and Basbaum, 2001; Lee, 1976).
A large number of peptides as isolated mu-conotoxins are known from Patent Application WO 02/07678 (University of Utah Research Foundation and Cognetix, Inc.). However, this document provides an ambiguous and at times misleading description of the peptides so that it is difficult to rely on its disclosures. For the large part, most of the peptides described therein appear to have been only identified by molecular biology techniques, by the isolation and cloning of DNA coding for mu-conotoxin peptides, translating and determining the toxin sequence. Reliance only on such techniques can cause errors, since in nature the active amino acid residues may result from posttranslational modification of the encoded peptide, some which can not be directly discovered from the nucleotide sequence.
Recently, Patent Application WO 2004/0099238 (The University of Queensland) also disclosed novel mu-conotoxin peptides and derivatives thereof with their use as neuronally active sodium channel inhibitors (antagonists), in assays and probes and also in the treatment of conditions involving pain, cancer, epilepsy and cardiovascular diseases. This application also disclosed the use of these novel mu-conotoxin peptides in radio-ligand binding assays (RLB). It will be appreciated by a skilled person in the art that these results do not imply any biological activity of the mu-conotoxins but only a binding effect since it is known from the literature that some compounds (including a conotoxin) bind to their channel/receptor site without any biological activity (Fainzilber et al., 1994; Shichor et al., 1996). Moreover, potency indicated in these binding experiments would not be relevant to inhibitory potency in vitro or in vivo, thus leaving the reader ignorant of any potential biological inhibitory potency. Furthermore, the only inhibitory activity (IC50) on the expressed VSSCs channels mentioned are superior to 1 to 3 μM thus suggesting an even higher concentration for use in ex vivo or in vivo preparation.
Thus novel compounds with potent and long-lasting biological activity for application as anesthetics which have a good safety profile, only low or no side effects and the possibility to retreat, whenever necessary are still needed.
This object has been achieved by providing novel mu-conotoxin peptides, a biologically active fragment thereof, a salt thereof, a combination thereof and/or variants thereof. The peptides of the invention, which present a long duration of effects, can be useful in the preparation of an anesthetic and in the treatment of a pain.